FIGURE 7.

D-box mutation suppresses human Mps1 interactions with Cdc20 or Cdh1 and inhibits polyubiquitination of Mps1. GFP-tagged Mps1 proteins (wild type or D-box mutant) were ectopically expressed in 293T cells and subsequently arrested at mitosis by nocodazole treatment or synchronized at late G₁ phase as described under “Experimental Procedures.” Cell cycle synchronization was confirmed by flow cytometry. Co-IP assays were performed with antibodies to Cdc20 or Cdh1 proteins to assess whether either protein associates with the Mps1 D-box mutant. A, Mps1 D-box mutant fails to co-precipitate with Cdc20 at mitosis. B, Mps1 D-box mutant fails to co-precipitate with Cdh1 at G₁ phase. C, mutation of the D-box inhibits polyubiquitination of Mps1. 293T cells were transiently co-transfected with equal amounts of wild type GFP-Mps1 or D-box-mut-Mps1 plasmids together with HA-tagged Cdc20 or Cdh1 and His₆-ubiquitin-containing plasmids. Expression levels of GFP-Mps1 WT or D-box mut and HA-tagged Cdc20 and Cdh1 proteins are shown in the right panel. Polyubiquitination of Mps1 wild type or D-box mutant proteins was assessed as described under “Experimental Procedures.” WB, Western blot.